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To investigate the effects of a neonatal low-protein diet on the number of macrophages in culture and the expression/production of proteins that regulate macrophage fusion in young and adult rats.Male Wistar rats (n = 18) were suckled by mothers fed diets containing 17 % protein (controls, C) or 8 % protein (undernourished, UN). All rats were fed a normal protein diet after weaning. Bronchoalveolar lavage was collected from 42-, 60- and 90-day-old rats. Alveolar macrophages were cultured for 4 days to assess the number of cells and the expression of cadherins, key proteins involved in macrophage fusion, by western blotting. IL-4 and IFN-γ levels in culture supernatants were measured by ELISA.Offspring from mothers fed a low-protein diet showed a lower body weight gain. The number of cells in cultured macrophages from UN was reduced at 42 and 60 days and increased at 90 days. IL-4 production was increased in the supernatants from UN group at 60 days but did not affect the expression of cadherins. IFN-γ production was increased in the supernatants from UN group at 42 and 60 days and reduced at 90 days.This study thus demonstrated that dietary restriction during lactation altered the number of alveolar macrophages in culture and the production of fusion proteins of offspring aged 42, 60 or 90 days but did not modify the expression of adhesion molecules important for the fusion of these cells.

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2-hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin-3 (Gal-3) protein.Incubation with okra RG-I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N-cadherin and alpha5 integrin subunit was reduced and that of the multifunctional carbohydrate-binding protein, Gal-3, at the cell membrane increased.These findings suggest that okra RG-I induces apoptosis in melanoma cells by interacting with Gal-3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur.

Ti-based biocompatible alloys are especially used for replacing failed hard tissue. Some of the most actively investigated materials for medical implants are the beta-Ti alloys, as they have a low elastic modulus (to inhibit bone resorption). They are alloyed with elements such as Nb, Ta, Zr, Mo, and Fe. We have prepared a new beta-Ti alloy that combines Ti with the non-toxic elements Ta and Mo using a vacuum arc-melting furnace and then annealed at 950 degrees C for one hour. The alloy was finally quenched in water at room temperature. The Ti-12Mo-5Ta alloy was characterised by X-ray diffraction, optical microscopy, SEM and EDS and found to have a body-centred-cubic structure (beta-type). It had a lower Young's modulus (about 74 GPa) than the classical alpha/beta Ti-6Al-4V alloy (120 GPa), while its Vickers hardness remained very high (about 303 HV). This makes it a good compromise for a use as a bone substitute. The cytocompatibility of samples of Ti-12Mo-5Ta and Ti-6Al-4V titanium alloys with various surface roughnesses was assessed in vitro using organotypic cultures of bone tissue and quantitative analyses of cell migration, proliferation and adhesion. Mechanically polished surfaces were prepared to produce unorientated residual polished grooves and cells grew to a particularly high density on the smoother Ti-12Mo-5Ta surface tested.

Biomaterials bearing functional groups can be important for designing colloidal drug carriers or optimising implants and medical devices. Particle dissolution or the wear of biomaterials can cause inflammation mediated by macrophages and fibroblasts. We have investigated the in vitro uptake of standard and functionalised (COO − or R3N + ) 0.4 m polystyrene (PS) beads by J774.2 macrophages and L929 fibroblasts. Both COO − and R3N + beads decreased the number of J774.2 cells after 48 h. Charged (especially R3N + ) but not standard beads are toxic for L929. An increase in cell size was correlated with cytotoxicity in every case. Both cell types produced increased IL6 secretion, measured by ELISA, when incubated for 24 h with standard PS or R3N + beads. Uptake of COO − and R3N + bead by J774.2 cells caused a dramatic increase in TNF release. Internalisation at 6 and 24 h was determined by measuring granularity by flow cytometry; it showed that L929 cells take up beads more slowly than J774.2 cells, but that they rapidly and avidly internalise R 3N + beads. Macrophages internalise roughly same amounts of all bead types. Cell morphology and intracellular bead arrangement change with the surface properties of the PS beads. Both cytochalasin D (2.5 M) and colchicine (10 M) inhibited standard bead uptake at 6 h by J774.2 and L929 cells. The uptake of charged beads was also decreased with cytochalasin D, but colchicine did not inhibit the uptake of carboxylated beads. We evidenced that the functional groups on the bead surface influence bead uptake and the subsequent cell reactions. Analysis of serum protein adsorption onto beads could well help identify the cellular mechanisms underlying these regulations. © 2003 Elsevier B.V. All rights reserved.

The cytotoxicity caused by the debris resulting from wear of prostheses can produce major damage to tissues around the implant. We have compared particle internalization by macrophages and fibroblasts in vitro and analyzed cell death. J774.2 macrophages and L929 fibroblasts were incubated with 0.43 and 2.81 microm alumina particles or 0.45 and 3.53 microm polystyrene (PS) beads. Incubation of J774.2 cells with alumina particles of both sizes and 0.5 and 1.0 mg/ml PS beads significantly decreased cell numbers in a particle concentration-dependent manner. L929 cells were not affected by lower concentrations of 0.43 microm alumina particles (which aggregate at high concentrations) and they internalized 0.45 microm PS beads without any decrease in cell numbers. Particles were more cytotoxic for macrophages than for fibroblasts. Particles caused the size of both types of cells to increase in correlation with cytotoxicity. Trypan blue exclusion and lactate dehydrogenase release showed cell membrane leakage for both types of cells incubated with PS beads for 24 h. Apoptosis was assessed by annexin V-FITC, propidium iodide staining and assay of caspase 3 activity. Macrophage death appeared to depend on both necrosis, caused mainly by 3.53 microm PS beads, and apoptosis, mainly due to 0.45 microm PS beads. The release of the inflammatory cytokine IL-6 appears to be nonlinearly correlated with cytotoxicity. Thus, the size of the internalized particles affects macrophages and fibroblasts differently, and the increase in cell size can be used as a preliminary criterion of particle cytotoxicity in vitro.

Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling.

We have previously shown that the adenylyl cyclase, which produces cyclic AMP (cAMP) in Swiss 3T3 cells, is activated by their attachment to a cellulose substratum (Cuprophan, CU). This substratum adsorbs vitronectin poorly, prevents cell spreading and causes them to aggregate. By contrast, cells spread out on polystyrene and contain low concentrations of cAMP. We have found that Connexin 43 (Cx 43) gap junction plaques are involved in this cell aggregation. MDL 12330 A, a specific inhibitor of adenylyl cyclase, prevented cell aggregation on CU and abolished Cx 43 channel clustering. But forskolin, a direct activator of adenylyl cyclase, and SBr cAMP, a cell-permeable analogue of cAMP, caused Cx 43 channel clustering in cells attached to polystyrene. Hence, Cx 43 channel clustering is regulated by cAMP in Swiss 3T3 cells. In addition, neither brefeldin A nor monensin (inhibitors of transit through the endoplasmic reticulum and Golgi apparatus), abolished Cx 43 channel clustering in cells aggregated on CU. Thus, the Cx 43 that form clusters in cells attached to CU are not dependent upon the trafficking of Cx 43 from intracellular storage sites, but are probably reorganised from the plasma membrane.

Purpose: Embolic events during carotid angioplasty are a challenging problem. This experimental study was undertaken to determine the embolic risk after each stage of carotid angioplasty procedure. Methods: Five ex vivo carotid artery balloon angioplasties were performed on fresh carotid specimens. The carotid specimens were obtained from five patients who underwent an internal carotid artery bypass for stenosis >75%. Before the endovascular maneuvers and after each stage of the procedures, the specimens were flushed with 20 mL of saline solution. Small particulate emboli (diameter

The processing of signals produced when cells contact biomaterials was examined. Of the several possible pathways, this study focuses on the amount of cAMP that accumulated in NIH 3T3 cells during the first 45 min after the cells contacted the bioincompatible membrane Cuprophan (CU) and the biocompatible membrane AN69. The cells that adhered to CU contained more cAMP than those that attached to AN69. This might be because the cells did not spread but remained rounded up under scanning electron microscopy. There was no increase in cAMP in the cells that did not adhere to CU. The cAMP-modulating agents, forskolin and isoproterenol, were used to assess the cAMP-generating capacity of adenylylcyclase in cells adhering to CU and AN69. This capacity was not affected by a high concentration (100 microM) of forskolin. Isoproterenol had no effect on the cAMP content of the cells, demonstrating that beta adrenergic receptors are not implicated in the activation of cAMP production by membranes. The bioincompatibility of CU seems to be responsible for the greater amount of cAMP in adherent cells, and this parameter could provide an index for assessing biocompatibility.

To investigate the effects of a neonatal low-protein diet on the morphology of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats.Male Wistar rats (n = 18) were suckled by mothers fed diets containing 17% protein (controls, C) or 8% protein (undernourished, UN). All rats were fed a normal protein diet after weaning. Muscles were removed from the legs of 42-, 60- and 90-day-old rats. Muscle cells were cultured to assess cell number, morphology and the expression of major proteins involved in myogenesis (Pax7, cadherins, β1 integrin, IL-4Rα and myogenin) by western blotting. IL-4 levels in culture supernatants were measured by ELISA.Offspring from mothers fed a low-protein diet showed a lower body weight gain. Cell number and myotube expansion were reduced in cultured muscle cells from UN, but the expression of myogenic marker proteins was unaltered.Dietary restriction during lactation had no impact on the synthesis of myogenic marker proteins, and myocyte differentiation occurred normally in the muscles of offspring aged 42, 60 or 90 days. Nevertheless, the number and morphology of the myotubes are altered.

The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-alpha) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24h on MHR-alpha-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-alpha secretion were studied. The results indicate that MHR-alpha coating inhibits the LPS-induced activation of macrophages.

Aging affects many motor functions, notably the spinal stretch reflexes and muscle spindle sensitivity. Spindle activation also depends on the elastic properties of the structures linked to the proprioceptive receptors. We have calculated a spindle efficacy index, SEI, for old rats. This index relates the spindle sensitivity, deduced from electroneurograms recording (ENG), to the passive stiffness of the muscle. Spindle sensitivity and passive incremental stiffness were calculated during ramp and hold stretches imposed on pseudo-isolated soleus muscles of control rats (aged 4 months, n = 12) and old rats (aged 24 months, n = 16). SEI were calculated for the dynamic and static phases of ramp (1–80 mm/s) and for hold (0.5–2 mm) stretches imposed at two reference lengths: length threshold for spindle afferents discharges, L n (neurogram length) and slack length, L s . The passive incremental stiffness was calculated from the peak and steady values of passive tension, measured under the stretch conditions used for the ENG recordings, and taking into account the muscle cross-sectional area. The pseudo-isolated soleus muscles were also stretched to establish the stress–strain relationship and to calculate muscle stiffness constant. The contralateral muscle was used to count muscle spindles and spindle fibers (ATPase staining) and immunostained to identify MyHC isoforms. L n and L s lengths were not significantly different in the control group, while L n was significantly greater than L s in old muscles. Under dynamic conditions, the SEI of old muscles was the same as in controls at L s , but it was significantly lower than in controls at L n due to increased passive incremental stiffness under the stretch conditions used to analyze the ENG. Under static conditions, the SEI of old muscles was significantly lower than control values at all the stretch amplitudes and threshold lengths tested, due to increased passive incremental stiffness and decreased spindle sensitivity at L s . The muscle stiffness constant values were greater in old muscles than in controls, confirming the changes in elastic properties under passive conditions due to aging. Aging also altered the intrafusal fibers: it increased the mean number of intrafusal fibers and the contents in the slow, neonatal and developmental isoforms intrafusal of MyHC have been modified. These structural modifications do not seem great enough to counteract the loss of the spindle sensitivity or the spindle efficacy under passive conditions and after the nerve was severed. However, they may help to maintain the spindle afferent message under natural conditions and under fusimotor control.

Controlling cell shape induced by cell–substrata interaction appears of prime importance to influence subsequent biological processes such as cell migration, proliferation, or apoptosis [1–3]. Cell shape and cytoskeletal organization may change through the transmission of mechanical stresses mediated by cell-surface integrins. Integrins are receptors composed of α and β subunits linked in a transmembrane heterodimer. Some of these mediate the adhesion of cells to Arg-Gly-Asp (RGD)-containing proteins such as vitronectin (VN) or fibronectin (FN), both of which play a major role in the attachment of cells plated out in the presence of fetal bovine serum (FBS). Adsorbed proteins maintaining their sites in conformational active structures will be recognized then by specific integrins. In this respect, cell culture treated polystyrene dishes (PS) which adsorb adhesive serum proteins without altering them favor cell spread and proliferation. Since the surface of the material may be considered as a matrix of chemical groups influencing cell-material contact, every monomer unit of the surface is a potential site for interaction with proteins and cells. Therefore, depending on their surface properties, biomaterials will adsorb more or less adhesive proteins and initiate different cell behaviors.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Spindle discharges are affected by muscle unloading, and changes in passive stiffness of the muscle–tendon unit may contribute to the changes in spindle solicitation. To test this hypothesis, we determined the spindle sensitivity from electroneurograms of the soleus nerve, and, concomitantly, we measured the incremental passive muscle tension. Both measurements were done from ramp and hold stretches imposed to the soleus muscle after the Achilles tendon was severed. The ratio between the spindle sensitivity and the passive stiffness gave a “spindle efficacy index” (SEI). The experiments were conducted on control rats (C, n = 12) and on rats that had undergone hindlimb unloading (HU, n = 12) for 21 days. The muscle threshold lengths for electroneurogram to discharge (neurogram length, Ln) and for detecting passive tension (slack length, Ls) were determined, and, when these lengths differed, the stretches were imposed at these two initial lengths. The contralateral muscles were used to count muscle spindles and spindle fibers (ATPase staining) and to identify MyHC isoforms by immunostaining. Ln and Ls values were identical for the C muscles, while after HU, Ln was significantly shorter than Ls, which indicated that spindle afferents were more sensitive since they discharged before any passive tension was developed by the soleus muscle. At Ln, spindle sensitivity and passive stiffness did not differ for C and HU muscles. Consequently, when calculated at this relatively short initial muscle length, the SEI was maintained (or even slightly increased) after HU. This held under dynamic conditions (ramp phase of the stretch) and under static conditions (hold phase of the stretch). At Ls, the dynamic and static incremental stiffness values increased significantly after HU. Under dynamic conditions, the spindle sensitivity also increased after HU but to a less degree than incremental stiffness, which led to a significant decrease in SEI. Under static conditions, the spindle sensitivity presented a high increase, and, consequently, SEI was not modified. These functional changes were associated with structural adaptations: HU did not alter the total number of muscle spindles, but the number of spindles containing three nuclear chain fibers increased significantly. The main change in intrafusal MyHC content concerned the slow type I MyHC isoform. In conclusion, after a period of muscle unloading, the spindle discharges were maintained or even enhanced in several experimental conditions. This may be due to a better transmission of the external stretch to muscle spindles through stiffer elastic structures but also to own muscle spindle adaptations which reinforce the spindle sensitivity, notably under static conditions.

Cells aggregate on an original cellulose substratum (CEL). This influences the signaling programs of adhering cells. CEL thus appears to be a suitable tool for studying the regulation of cell-substratum and cell-cell interactions.

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

Biomaterials used in some bioreactors are porous and exposed to normal and tangential flow of physiological fluid. Flow-induced forces may influence the morphological and biochemical responses of cells adhering to these materials. The objective of this work is to examine the capacity of mechanical stress to cause changes in cell morphology via the cAMP pathway (cyclic adenosine monophosphate). This second messenger is known to modulate cell morphology in static conditions. In classical flow devices, cells are submitted to only tangential stresses. We designed a new flow system, a Hele-Shaw cell with a porous bottom wall, in order to take into account the influence of a transmural pressure. This flow chamber allows to follow up continuously the shape changes of cells that are adherent to a porous biomaterial (polyacrylonitrile) and are exposed to controlled levels of shear stress or transmural pressure. Mouse Swiss 3T3 fibroblasts exposed to a 1.1-Pa shear stress, as well as those exposed to a 84-mm Hg transmural pressure, round up (up to 50%) in a few minutes. If the cAMP pathway is inhibited when a mechanical stress is applied, cell rounding is significantly prevented. These observations suggest that flow-induced cell shape changes are cAMP-dependent. This conclusion is supported by an increased cAMP accumulation measured in cells under mechanical stress when compared to static experiments. Our in vitro flow system is thus useful to study the influence of transmural pressure or shear stress on the early morphological and biochemical responses of cells in contact with a biomaterial.

We have examined the link between the aggregation or spreading of cells adhering to substrata of differing biocompatibility and activation of the cyclic AMP (cAMP) pathway. We compared the rate at which the Mouse Swiss 3T3 fibroblasts attached to Cuprophan (CU), AN69 and a control plastic in the presence and absence of foetal calf serum (FCS). Serum had no effect on the kinetics of cell attachment to CU or AN69. Cells incubated in culture medium containing 10% FCS aggregated on CU, whereas they spread on AN69 and plastic. Aggregated cells contained significantly higher concentrations of cAMP than cells spreading, and aggregation was prevented by treatment with miconazole, an inhibitor of adenylyl cyclase. cAMP-dependent cell aggregation occurred on all three substrata in serum-free medium, suggesting that proteins adsorbed onto AN69 and plastic in the presence of serum helped protect the cells. Far less serum protein was adsorbed onto CU than onto AN69 or plastic, consistent with the similar increases in cAMP in cells attached to CU with or without serum.

Using a fetal rat liver cell bioassay, serum erythropoietic activity was tested in nonsuckled newborn rats comparatively to 21-day-old rat fetuses at time 0 or after 2 h of surviving. The number of erythroid colonies (CFUe) is significantly greater in cultures with neonatal and fetal rat serum after 2 h of surviving than in cultures with fetal rat serum sampled at time 0. These results suggest a release of erythropoietin or erythropoietin-like substances in response to a short hypoxic phase following the parting of the fetus with the mother and preceding the autonomous oxygenation of the newborn by breathing.

Fetal hepatic erythron misfunction, experimentally induced at the end of intrauterine life of the rat either by corticosteroid deprivation (fetal hypophysectomy following maternal adrenalectomy) or, on the contrary, by corticosteroid excess (repeated maternal stress), leads to an increase in serum erythropoietic activity detected with an in vitro fetal liver bioassay. This increase in serum erythropoietin-like substance may be related with the premature onset of bone marrow red cell formation observed under similar experimental conditions.